Arginine-specific cleavage is the primary method used to prepare lysine-rich histone proteins in bottom-up proteomics. As the Arg-C enzyme has demonstrated suboptimal specificity, cleavage at the carboxyl side of arginine residues is typically achieved through the chemical derivatization of lysines followed by trypsin digestion. Recent improvements in proteolytic enzymes are reflected in the introduction of Arg-C Ultra, a recombinant proteinase with a substantially improved digestion specificity. Here, using mammalian histone extract, we demonstrate that Arg-C Ultra facilitates histone preparation for LC-MS/MS. We show the performance of Arg-C Ultra in terms of digestion specificity, number of modified forms identified, and yield of quantitative information compared with Arg-C and trypsin digestion combined with chemical derivatization with trimethylacetic anhydride. Importantly, we show that chemical derivatization at the peptide level, i.e., after Arg-C Ultra digestion, is still necessary to improve the quantification of short histone peptidoforms as well as positional isomers.

• MeSH
• Arginine metabolism   chemistry   MeSH
• Chromatography, Liquid methods   MeSH
• Histones * chemistry   metabolism   isolation & purification   MeSH
• Liquid Chromatography-Mass Spectrometry MeSH
• Humans MeSH
• Tandem Mass Spectrometry * methods   MeSH
• Trypsin metabolism   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Arginine MeSH
• Histones * MeSH
• Trypsin MeSH

BACKGROUND: Obesity is a major health burden. Preadipocytes proliferate and differentiate in mature adipocytes in the adipogenic process, which could be a potential therapeutic approach for obesity. Deficiency of SIRT6, a stress-responsive protein deacetylase and mono-ADP ribosyltransferase enzyme, blocks adipogenesis. Mutants of SIRT6 (N308K/A313S) were recently linked to the in the long lifespan Ashkenazi Jews. In this study, we aimed to clarify how these new centenarian-associated SIRT6 genetic variants affect adipogenesis at the transcriptional and epigenetic level. METHODS: We analyzed the role of SIRT6 wild-type (WT) or SIRT6 centenarian-associated mutant (N308K/A313S) overexpression in adipogenesis, by creating stably transduced preadipocyte cell lines using lentivirus on the 3T3-L1 model. Histone post-translational modifications (PTM: acetylation, methylation) and transcriptomic changes were analyzed by mass spectrometry (LC-MS/MS) and RNA-Seq, respectively, in 3T3-L1 adipocytes. In addition, the adipogenic process and related signaling pathways were investigated by bioinformatics and biochemical approaches. RESULTS: Overexpression of centenarian-associated SIRT6 mutant increased adipogenic differentiation to a similar extent compared to the WT form. However, it triggered distinct histone PTM profiles in mature adipocytes, with significantly higher acetylation levels, and activated divergent transcriptional programs, including those dependent on signaling related to the sympathetic innervation and to PI3K pathway. 3T3-L1 mature adipocytes overexpressing SIRT6 N308K/A313S displayed increased insulin sensitivity in a neuropeptide Y (NPY)-dependent manner. CONCLUSIONS: SIRT6 N308K/A313S overexpression in mature adipocytes ameliorated glucose sensitivity and impacted sympathetic innervation signaling. These findings highlight the importance of targeting SIRT6 enzymatic activities to regulate the co-morbidities associated with obesity.

• Keywords
• Adipogenesis, Epigenetics, Histones, Obesity, SIRT6,
• MeSH
• Adipogenesis * genetics   MeSH
• 3T3-L1 Cells * MeSH
• Epigenesis, Genetic * genetics   MeSH
• Histones metabolism   genetics   MeSH
• Humans MeSH
• Mutation MeSH
• Mice MeSH
• Obesity genetics   metabolism   MeSH
• Protein Processing, Post-Translational genetics   MeSH
• Sirtuins * genetics   metabolism   MeSH
• Adipocytes * metabolism   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Histones MeSH
• SIRT6 protein, human MeSH Browser
• Sirt6 protein, mouse MeSH Browser
• Sirtuins * MeSH

Histone deacetylases (HDACs) target acetylated lysine residues in histone and non-histone proteins. HDACs are implicated in the regulation of genomic stability, cell cycle, cell death and differentiation and thus critically involved in tumorigenesis. Further, HDACs regulate T-cell development and HDAC inhibitors (HDACis) have been approved for clinical use in some T-cell malignancies. Still, the exact targets and mechanisms of HDAC inhibition in cancer are understudied. We isolated tumor cell lines from a transgenic mouse model of anaplastic large cell lymphoma (ALCL), a rare T-cell lymphoma, and abrogated HDAC activity by treatment with the HDACis Vorinostat and Entinostat or Cre-mediated deletion of Hdac1. Changes in overall protein expression as well as histone and protein acetylation were measured following Hdac1 deletion or pharmacological inhibition using label-free liquid chromatography mass spectrometry (LC-MS/MS). We found changes in overall protein abundance and increased acetylation of histones and non-histone proteins, many of which were newly discovered and associated with major metabolic and DNA damage pathways. For non-histone acetylation, we mapped a total of 1204 acetylated peptides corresponding to 603 proteins, including chromatin modifying proteins and transcription factors. Hyperacetylated proteins were involved in processes such as transcription, RNA metabolism and DNA damage repair (DDR). The DDR pathway was majorly affected by hyperacetylation following HDAC inhibition. This included acetylation of H2AX, PARP1 and previously unrecognized acetylation sites in TP53BP1. Our data provide a comprehensive view of the targets of HDAC inhibition in malignant T cells with general applicability and could have translational impact for the treatment of ALCL with HDACis alone or in combination therapies.

• Keywords
• ALCL, MS-275, SAHA, acetylomics, anaplastic large cell lymphoma, entinostat, histone deacetylase inhibitors, histone deacetylases, proteomics, vorinostat,
• MeSH
• Acetylation MeSH
• Lymphoma, Large-Cell, Anaplastic * drug therapy   MeSH
• Chromatography, Liquid MeSH
• Histone Deacetylases * metabolism   MeSH
• Histones metabolism   MeSH
• Hydroxamic Acids pharmacology   MeSH
• Mice MeSH
• Tandem Mass Spectrometry MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Histone Deacetylases * MeSH
• Histones MeSH
• Hydroxamic Acids MeSH

BACKGROUND: Variants of linker histone H1 are tissue-specific and are responsible for chromatin compaction accompanying cell differentiation, mitotic chromosome condensation, and apoptosis. Heterochromatinization, as the main feature of these processes, is also associated with pronounced trimethylation of histones H3 at the lysine 9 position (H3K9me3). METHODS: By confocal microscopy, we analyzed cell cycle-dependent levels and distribution of phosphorylated histone H1 (H1ph) and H3K9me3. By mass spectrometry, we studied post-translational modifications of linker histones. RESULTS: Phosphorylated histone H1, similarly to H3K9me3, has a comparable level in the G1, S, and G2 phases of the cell cycle. A high density of phosphorylated H1 was inside nucleoli of mouse embryonic stem cells (ESCs). H1ph was also abundant in prophase and prometaphase, while H1ph was absent in anaphase and telophase. H3K9me3 surrounded chromosomal DNA in telophase. This histone modification was barely detectable in the early phases of mitosis. Mass spectrometry revealed several ESC-specific phosphorylation sites of H1. HDAC1 depletion did not change H1 acetylation but potentiated phosphorylation of H1.2/H1.3 and H1.4 at serine 38 positions. CONCLUSIONS: Differences in the level and distribution of H1ph and H3K9me3 were revealed during mitotic phases. ESC-specific phosphorylation sites were identified in a linker histone.

• Keywords
• chromatin, epigenetic, histone H1, histone H3, mass spectrometry, nucleolus,
• Publication type
• Journal Article MeSH

Histone post-translational modifications (hPTMs) are epigenetic marks that strongly affect numerous processes, including cell cycling and protein interactions. They have been studied by both antibody- and MS-based methods for years, but the analyses are still challenging, mainly because of the diversity of histones and their modifications arising from high contents of reactive amine groups in their amino acid sequences. Here, we introduce use of trimethylacetic anhydride (TMA) as a new reagent for efficient histone derivatization, which is a requirement for bottom-up proteomic hPTM analysis. TMA can derivatize unmodified amine groups of lysine residues and amine groups generated at peptide N-termini by trypsin digestion. The derivatization is facilitated by microwave irradiation, which also reduces incubation times to minutes. We demonstrate that histone derivatization with TMA reliably provides high yields of fully derivatized peptides and thus is an effective alternative to conventional methods. TMA afforded more than 98% and 99% labeling efficiencies for histones H4 and H3, respectively, thereby enabling accurate quantification of peptide forms. Trimethylacetylation substantially improves chromatographic separation of peptide forms, which is essential for direct quantification based on signals extracted from MS1 data. For this purpose, software widely applied by the proteomics community can be used without additional computational development. Thorough comparison with widely applied propionylation highlights the advantages of TMA-based histone derivatization for monitoring hPTMs in biological samples.

• Keywords
• bottom–up proteomics, chemical derivatization, histone post-translational modifications, microwave irradiation, trimethylacetic anhydride,
• MeSH
• Acetic Anhydrides chemistry   MeSH
• Acetylation MeSH
• Chromatography, Liquid MeSH
• Histones chemistry   MeSH
• Mice MeSH
• Cell Line, Tumor MeSH
• Protein Processing, Post-Translational MeSH
• Tandem Mass Spectrometry MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Acetic Anhydrides MeSH
• Histones MeSH

A high degree of developmental plasticity enables plants to adapt to continuous, often unfavorable and unpredictable changes in their environment. At the molecular level, adaptive advantages for plants are primarily provided by epigenetic machinery including DNA methylation, histone modifications, and the activity of noncoding RNA molecules. Using a mass spectrometry-based proteomic approach, we examined the levels of acetylated histone peptide forms in Arabidopsis plants with a loss of function of histone deacetylase 6 (HDA6), and in plants germinated in the presence of HDA inhibitors trichostatin A (TSA) and sodium butyrate (NaB). Our analyses revealed particular lysine sites at histone sequences targeted by the HDA6 enzyme, and by TSA- and NaB-sensitive HDAs. Compared with plants exposed to drugs, more dramatic changes in the overall profiles of histone post-translational modifications were identified in hda6 mutants. However, loss of HDA6 was not sufficient by itself to induce hyperacetylation to the maximum degree, implying complementary activities of other HDAs. In contrast to hda6 mutants that did not exhibit any obvious phenotypic defects, the phenotypes of seedlings exposed to HDA inhibitors were markedly affected, showing that the effect of these drugs on early plant development is not limited to the modulation of histone acetylation levels.

• Keywords
• Arabidopsis thaliana, epigenetics, histone, mass spectrometry, post-translational modifications, sodium butyrate, trichostatin A,
• MeSH
• Arabidopsis genetics   growth & development   MeSH
• Histone Deacetylases genetics   MeSH
• Histone Code drug effects   genetics   MeSH
• Histone Deacetylase Inhibitors pharmacology   MeSH
• Germination genetics   MeSH
• Butyric Acid pharmacology   MeSH
• Hydroxamic Acids pharmacology   MeSH
• DNA Methylation drug effects   MeSH
• Arabidopsis Proteins antagonists & inhibitors   genetics   MeSH
• Proteomics * MeSH
• Gene Expression Regulation, Plant MeSH
• Seedlings drug effects   genetics   MeSH
• Gene Silencing MeSH
• Plant Development drug effects   genetics   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• AT5G63110 protein, Arabidopsis MeSH Browser
• Histone Deacetylases MeSH
• Histone Deacetylase Inhibitors MeSH
• Butyric Acid MeSH
• Hydroxamic Acids MeSH
• Arabidopsis Proteins MeSH
• trichostatin A MeSH Browser

The incorporation of histone H3 with an acetylated lysine 56 (H3K56ac) into the nucleosome is important for chromatin remodeling and serves as a marker of new nucleosomes during DNA replication and repair in yeast. However, in human cells, the level of H3K56ac is greatly reduced, and its role during the cell cycle is controversial. Our aim was to determine the potential of H3K56ac to regulate cell cycle progression in different human cell lines. A significant increase in the number of H3K56ac foci, but not in H3K56ac protein levels, was observed during the S and G2 phases in cancer cell lines, but was not observed in embryonic stem cell lines. Despite this increase, the H3K56ac signal was not present in late replication chromatin, and H3K56ac protein levels did not decrease after the inhibition of DNA replication. H3K56ac was not tightly associated with the chromatin and was primarily localized to active chromatin regions. Our results support the role of H3K56ac in transcriptionally active chromatin areas but do not confirm H3K56ac as a marker of newly synthetized nucleosomes in DNA replication.

• Keywords
• Cell cycle, Chromatin, DNA replication, H3K56ac, Mammalian cells, Nucleosome,
• MeSH
• Cell Cycle genetics   physiology   MeSH
• Chromatin metabolism   MeSH
• G2 Phase genetics   MeSH
• Histones metabolism   MeSH
• HL-60 Cells MeSH
• Mass Spectrometry MeSH
• Humans MeSH
• Nucleosomes metabolism   MeSH
• DNA Replication genetics   physiology   MeSH
• S Phase genetics   MeSH
• Saccharomyces cerevisiae Proteins genetics   metabolism   MeSH
• Saccharomyces cerevisiae genetics   metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Chromatin MeSH
• Histones MeSH
• Nucleosomes MeSH
• Saccharomyces cerevisiae Proteins MeSH